The tetrazole analogue of the auxin indole-3-acetic acid binds

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The tetrazole analogue of the auxin indole-3-acetic acid binds preferentially to TIR1 and not AFB5. Mussa Quareshy, Justyna Prusinska, Martin Kieffer, Kosuke Fukui, Alonso J. Pardal, Silke Lehmann, Patrick Schafer, Charo I. del Genio, Stefan Kepinski, Kenichiro hayashi, Andrew Marsh, and Richard M. Napier ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00527 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018

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ACS Chemical Biology

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The tetrazole analogue of the auxin indole-3-acetic acid

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binds preferentially to TIR1 and not AFB5.

3

Mussa Quareshy 1†, Justyna Prusinska 1, Martin Kieffer 2, Kosuke Fukui 3, Alonso J. Pardal 1,

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Silke Lehmann 1,5, Patrick Schafer 1,5, Charo I. del Genio 1, Stefan Kepinski 2, Kenichiro

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Hayashi 3, Andrew Marsh 4, Richard M. Napier 1†

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1

School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK

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2

Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT

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3

Department of Biochemistry, Okayama University of Science, 1-1 Ridaicho, Kita-ku, Okayama-shi

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Okayama, JP 700-0005

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4

Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK

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5

Warwick Integrative Synthetic Biology Centre

12

†

Corresponding authors:

13

[email protected]; [email protected]

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Highlights: We apply a functional group swap to auxin yielding an active auxin that also confers

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selectivity between 2 members of the auxin receptor family.

16 17

Key words: Auxin, bioisostere, herbicides, tetrazole, synthetic herbicides, rationale, drug-design,

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SAR, target selectivity.

19 20

Who did what: MQ came up with the concept of iMT, performed most of the experiments including

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synthesis of iMT in AM’s lab with AM’s guidance in designing the synthesis. JP performed the SPR

22

SCK assays. MK performed the pull-down assays and SK provided the tir1-1 knockout lines. KH and

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KF synthesised the 4,5 and 6 Cl iMT analogues as well as additional iMT, performed the DR5::GUS

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and DII::VENUS reporter assays. CIDG performed the curve fitting analysis to derive the primary

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root growth IC50 values. AJP, SL and PS performed the protoplast reporter assays and AJP did the

26

qPCR. RN helped to develop the concept of iMT and was involved in the design of lead optimisation.

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MQ and RN wrote the manuscript with contributions from all the other authors.

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Abstract

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Auxin is considered one of the cardinal hormones in plant growth and development. It regulates a

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wide range of processes throughout the plant. Synthetic auxins exploit the auxin-signalling pathway

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and are valuable as herbicidal agrochemicals. Currently, despite a diversity of chemical scaffolds all

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synthetic auxins have a carboxylic acid as the active core group. By applying bio-isosteric

34

replacement we discovered that indole-3-tetrazole was active by surface plasmon resonance (SPR)

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spectrometry, showing that the tetrazole could initiate assembly of the TIR1 auxin co-receptor

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complex. We then tested the tetrazole’s efficacy in a range of whole plant physiological assays and in

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protoplast reporter assays which all confirmed auxin activity, albeit rather weak. We then tested

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indole-3-tetrazole against the AFB5 homologue of TIR1, finding that binding was selective against

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TIR1, absent with AFB5. The kinetics of binding to TIR1 are contrasted to those for the herbicide

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picloram, which shows the opposite receptor preference as it binds to AFB5 with far greater affinity

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than to TIR1. The basis of the preference of indole-3-tetrazole for TIR1 was revealed to be a single

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residue substitution using molecular docking, and assays using tir1 and afb5 mutant lines confirmed

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selectivity in vivo. Given the potential that a TIR1-selective auxin might have for unmasking receptor-

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specific actions, we followed a rational design, lead optimisation campaign and a set of chlorinated

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indole-3-tetrazoles was synthesised. Improved affinity for TIR1 and the preference for binding to

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TIR1 was maintained for 4- and 6-chloroindole-3-tetrazoles, coupled with improved efficacy in vivo.

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This work expands the range of auxin chemistry for the design of receptor-selective synthetic auxins.

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Introduction

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Auxin (indole-3-acetic acid; IAA (1) Figure 1) regulates diverse developmental processes including

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cell elongation, cell division, tropic responses, lateral rooting and branching, and synthetic auxins are

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an important class of selective herbicides. The best-studied auxin receptor is the F-box protein

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Transport Inhibitor Resistant 1 1. A wealth of experimentation has shown that TIR1 is the F-box

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component of a Skp, Cullin, F-box (SCF) type E3 ubiquitin ligase complex SCFTIR1, although it

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wasn’t until seminal work by two groups

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The TIR1 family includes five additional Auxin F-Box proteins (AFBs) in Arabidopsis and activity

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within this family has been shown to be largely redundant 4, with some notable exceptions. Root

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architecture responses to nitrate levels appear to be mediated through AFB3

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shown to be the dominant site of action for the picolinate herbicides 7 8.

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The mechanism of auxin action is coordinated through transcriptional regulation and this has been

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reviewed extensively

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proteins, together with co-repressor (TOPLESS) proteins repress genes targeted by the Auxin

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Response Factor (ARF) transcriptional activators. As concentrations rise, auxin binds to TIR1

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creating a high-affinity surface for recruitment of the AUX/IAA co-receptor. The assembly of this

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SCFTIR1co-receptor complex has been used for an auxin binding assay using surface plasmon

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resonance spectrometry 8, 12 . Assembly of the complex in vivo leads to ubiquination of the AUX/IAAs

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and consequent degradation in the proteasome. The consequent reduction in concentrations of

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AUX/IAA proteins releases the ARFs, allowing transcription to commence. The TIR1 crystal

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structure

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and radiolabel and SPR binding experiments illustrated that the wide dynamic range of responses to

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auxin may, in part, be accounted for by the range of affinities measured for different co-receptor

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complexes 12, 14, 15.

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The diversity of receptors suggests some differentiation of activity as well as dose dependence

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It was of interest to pursue the selectivity found for picolinate herbicides and investigate the

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possibility of receptor sub-class-specific ligands 16. We have explored the tetrazole functional group

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as a bioisostere (a chemical mimetic that sustains biologically activity) of carboxylic acids

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shown that indole-3-methyl tetrazole (compound 5, Figure 1; iMT) not only works as a weak auxin,

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but that it binds selectively to TIR1, and not to AFB5. Rational design was shown to improve its

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affinity for TIR1 without changing this selectivity.

13

2, 3 9 10, 11

2, 3

that TIR1 was shown to be the major auxin receptor.

5 6

and AFB5 has been

. At low auxin concentrations, AUX/IAA transcriptional repressor

provided the paradigm for IAA acting as molecular glue between TIR1 and AUX/IAA,


5, 6 14

17, 18

.

and


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Results and Discussion

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Modelling alternative ligands for TIR1

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Using the TIR1 crystal structure with IAA bound (PDB file 2P1P) we inspected the atomic distances

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from the carboxylic oxygens of IAA to nearby residues and noted distances of 3.47Å to Arg403 and

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4.52Å to Arg436 (Figure 2A). This indicated some exploitable space in this region and so we modelled

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iMT (Figure 1) in the TIR1 site using the structure builder feature in Chimera

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comparison of IAA with iMT (Figure 2 A & B) show the indole rings to be superimposable, with the

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tetrazole group extending past the position of the carboxylic acid in IAA, further down into the

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pocket. In our model the atomic distances were 1.89Å to Arg403 and 2.34Å to Arg436. Furthermore,

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the proximity of the Ser438 residue was also considered a probable hydrogen bonding partner for iMT

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19

. Side-by-side

(Figure 2B).

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Docking iMT into TIR1

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Docking algorithms use more robust molecular force field calculations than a structure mutation in

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Chimera and so iMT and IAA were docked using the Vina algorithm

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pocket of TIR1 using the coordinates from crystal structure 2P1Q (TIR1 with ligand and IAA7 degron

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bound, but removed for docking). We reasoned that 2P1Q would be the most appropriate template for

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docking in order to simulate the active state. Although there are no gross conformational changes

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during binding

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Docking predicted that the indole ring of bound iMT (scoring function -8.2 kcal/mol) would be

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superimposed onto that for docked (scoring function -8.1 kcal/mol) and crystallographic IAA (Figure

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2C), with a slight difference between alignments of the tetrazole and carboxylic acid. Interatomic

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distances between the tetrazole and neighbouring arginines show 3 hydrogen bond donors within

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range (Figure 2D). Thus in silico docking predicted that iMT would bind to TIR1, making it a non-

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carboxylic acid auxin.

13

20 21

into the auxin-binding

, there are subtle side group moves implied from the different crystal datasets.

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iMT binds TIR1 in vitro and acts as an auxin in vivo

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iMT was synthesised in a single step reaction from indole-3-acetonitrile with the addition of azide in a

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cycloaddition reaction (Supporting Information Scheme 1; physicochemical properties are also

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presented in Supporting Information Table 1). It was tested for binding to TIR1 using SPR. When

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mixed with purified TIR1, iMT supported TIR1 co-receptor assembly on the SPR chip with an

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activity of 18% relative to IAA (both at 50 µM; Figure 3A). We then used SPR to screen a selection


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of other aromatic tetrazoles, including (2-(naphthalen-1-yl)tetrazole), the tetrazole equivalent of 1-

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NAA (11, Supporting Information Figure 1). None of this collection of compounds bound to TIR1 or

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AFB5, consistent with a lack of auxin activity in previous whole plant assays 22 23.

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It is notable that the SPR binding signal from iMT plateaued rapidly on both association and

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dissociation phases (Figure 3A) unlike that for IAA, suggesting more rapid kinetics. Single cycle

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kinetics recorded a KD of 210 µM for iMT, compared to 5 µM for IAA (Table 1). The lower affinity

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for iMT is contributed by a 15-fold faster off-rate constant than for IAA, and an almost 3-fold slower

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on-rate constant.

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The SPR assays are performed with TIR1 expressed in insect cells. In order to check activity with

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plant-expressed TIR1, we tested the efficacy of iMT in pull-down assays with FLAG-tagged AtTIR1

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expressed in Nicotiana benthamiana plus IAA7 peptide 24. We observed a weak response compared to

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IAA (Figure 3C), which is consistent with the SPR data on kinetic rates.

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iMT lead optimisation

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Whilst iMT has been shown to be an active auxin, it is considerably weaker than IAA and many other

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synthetic auxins, and so rational design was applied to improve its activity. Past structure-activity

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relationship studies for auxins have shown that the addition of chlorine at the 4 or 6 positions of IAA

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yields potent auxins

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Therefore, to start lead optimisation of iMT we synthesised the corresponding 4-, 5- and 6-

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monochlorinated analogues (Supporting Information Scheme 2). Binding analysis using SPR showed

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that 4-Cl-iMT did have enhanced binding to TIR1 (Figure 3A; Table 1. Addition of chlorine at the 6

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position did not improve or reduce affinity and addition of Cl at the 5 position significantly decreased

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affinity for TIR1 and reduced activity in vivo (Table 2).

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Activity in planta.

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Having established that iMT was active as an auxin in receptor binding assays, it was necessary to test

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whether or not binding translated into auxin activity in planta.

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seedlings were grown on agar containing the test compound, iMT induced GUS activity at 50 µM,

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compared to IAA which gave a signal at 1 µM (Figure 4A). We followed this up with the auxin

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reporter DII::VENUS line

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presence of IAA. Again, iMT showed an auxin-like response, but reduced compared to that of IAA

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(Supporting Information Figure 2). Supporting these data, we treated 6-day-old DR5::GFP reporter

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line seedlings with IAA or iMT and recorded the GFP signal from primary root tips after 2 and 24

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hours (Figure 4B). After 2 hours only IAA induced the characteristic increase in GFP signal in the

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epidermis, steele and the lateral root cap

25, 26 27

. We also envisaged the chlorines might improve uptake properties

28

27

.

When DR5::GUS Arabidopsis

which showed a characteristic marked decrease in YFP signal in the

29

. After 24 hours iMT-treated roots also showed an



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enhanced GFP signal in cells types characteristic of auxin responses, although it remained weaker

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than the IAA-induced signal (Figure 4B). We also noted responses to iMT analogues 4-Cl-iMT and 6-

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Cl-iMT in the DR5::GUS assays (Figure 4A), in the DII:VENUS reporter assay (Supporting

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Information Figure 2) and in the DR5::GFP assay at 24 hours, especially for 4-Cl-iMT (Figure 4B).

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In order to reveal the timescale of responsiveness in live cells, we used a protoplast transient reporter

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assay

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4C; Supporting Information Figure 3). A series of doses of IAA or 4Cl-iMT were applied in parallel

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and the results indicated extended response lag times for iMT analogues compared to IAA, with

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responses becoming evident only after about 2 hours. This is consistent with the DR5::GFP data

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above (showing no response at 2 h) and might represent poor uptake kinetics for iMT and its

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analogues. Nevertheless, a clear, auxin-dependent signal is generated as 4Cl-iMT accumulates.

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Root growth assays quantify auxin activity

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The genetic reporter assays provided evidence that iMT was an active auxin in vivo and showed

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uptake of the compound into Arabidopsis roots and into protoplasts. However, genetic reporter assays

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are difficult to quantify and so we conducted dose-response assays using seedling root growth

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inhibition to evaluate IC50 values for each compound (Table 2). Defining the IC50 value as the

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concentration of compound needed to reduce primary root growth to 50% of the length without

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treatment, we obtained an IC50 value of 46 µM (±3) for iMT compared to 41 nM (±7) for IAA in Col-

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0. We observed complete root growth inhibition at 300 µM for iMT compared with 11 µM for IAA

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(Supporting Information Figure 4). The 1000-fold difference in activity in vivo is greater than the

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difference in affinity observed for receptor binding (approximately 30-fold, Figure 3C). We noted

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above that the response to iMT was slower than for IAA (Figure 4C), and these extended IC50 values

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also suggest that uptake of iMT by plant cells is impaired affecting potency. Nevertheless, in the same

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assay plates we observed increased lateral root density after iMT treatment, another characteristic

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auxin response (Figure 5), and so iMT is acting positively as an auxin, and not by interfering with

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selective elements of auxin physiology as reported for some other small molecules such as cis-

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cinnamic acid and 3,4-(methylenedioxy)cinnamic acid 31, 32.

30

adapted for use with auxin-sensitive promoters and automated to record continually (Figure

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iMT does not bind AFB5 and this can be explained using molecular docking

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In addition to testing iMT for binding to TIR1, we investigated binding to AFB5 (Figure 3B).

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Somewhat surprisingly, we noted that iMT did not induce AFB5 co-receptor assembly, revealing a

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selectivity towards TIR1. This is the opposite selectivity to that found for picloram and the 6-

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arylpicolinate DAS534, which bind preferentially to AFB5 7 8.


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In order to investigate the basis of iMT’s failure to bind to AFB5 we identified the residues lining the

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auxin-binding pocket of each receptor

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6A). There are two key changes; His78 becomes an arginine, and Ser438 becomes an alanine in

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AFB5. A change of histidine to arginine represents an increase in residue size and polarity, whilst a

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change of serine to an alanine is a decrease in polarity, an increase in hydrophobicity and loss of a key

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hydrogen bond acceptor. To understand how these changes might result in binding selectivity we

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used the crystal structure of TIR1 (PDB file 2P1Q) and the homology model built for AFB5 from this

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template

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contributing factor to iMT receptor selectivity. IAA docked into the AFB5 pocket in a favourable

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pose, resembling that for TIR1, such that the aromatic ring is surrounded with hydrophobic residues

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parallel to the base of the pocket, and the carboxylic acid is orientated towards arginine residues at the

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base of the pocket, and towards the centre of the protein. Docking iMT into the AFB5 pocket

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suggested that the increased steric bulk of the arginine displaced the tetrazole group upwards, with

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this polar group now imposing on space previously taken by the alpha-carbon of IAA, and tilting the

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pose of the indole ring with respect to the base of the pocket. Such a pose for iMT would be likely to

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reduce favourable interactions with the AUX/IAA degron by perturbing the hydrophobic interactions

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of auxin with the WPPV motif 13. Binding analysis of the monochlorinated iMT analogues revealed

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that binding remained selective for TIR1 with no binding to AFB5 (Figure 3; Table 1)

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Mutant Arabidopsis lines confirm that receptor specificity is maintained in vivo.

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In order to confirm that iMT is selective for TIR1 in planta, loss of function mutant lines of A.

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thaliana were evaluated in the root growth assay using dose-response experiments (Supporting

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Information Figure 4) In the tir1-1 line we observed a shift in IC50 from 46 µM for iMT to 90 µM

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(Table 2), and even at the highest dose of 300 µM primary root growth was not totally inhibited. With

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afb5-5 we observed no change in the IC50 value compared to wild type, consistent with the specificity

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seen for iMT and TIR1 in vitro. Specificity is also apparent for the lateral root growth trait (Figure 5),

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with the pattern of responsiveness to iMT being identical between Col-0 and afb5-5, but distinct from

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tir1-1. Tests were extended to quantitative RT-PCR to evaluate responsiveness using widely used

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auxin-responsive genes (Figure 4D). Treatment with IAA (1 µM) and 4-Cl-iMT (10 µM) gave similar

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auxin-like responses in Col-0, but the response to 4-Cl-iMT was absent in tir1-1 for all three reporter

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genes, consistent with binding selectivity of 4-Cl-iMT for TIR1. The reporters GH3.3 and GH3.1

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gave partial responses to 4-Cl-iMT in afb5-5, whilst reporter IAA5 responded as for IAA, as

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anticipated for a line with a functional TIR1. Selectivity for TIR1 was also shown to be maintained

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with lateral root density in the tir1-1 line being significantly reduced compared to Col-0 and afb5-5 at

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the active higher dose rates (Figure 5,Table 2). Picloram demonstrated its inverse selectivity and

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preference for AFB5 (Figure 5A).

12

33

and aligned complementary sequences from AFB5 (Figure

for docking in silico (Figure 6B to G). The His438Arg change is the most likely



223 224

Discussion

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We have demonstrated that isosteric replacement of the carboxylic acid on IAA with a tetrazole yields

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an active auxin that binds to the auxin receptor TIR1. Further, this isosteric change confers on iMT

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selectivity for TIR1 which introduces the first auxin that does not engage with the receptor homologue

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AFB5. Isosteric replacements have been reported for auxin previously, including the report of weak

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activities in the Avena coleoptile extension and pea split epicotyl assays 22, 23, but never pursued. The

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tetrazoles of 1-naphthylacetic acid and 2,4-dichlorophenoxyacetic acid were found to be far weaker

231

than iMT, as were other bioisosteres evaluated at the same time. Given the weak activity, perhaps it

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is not surprising that no further interest has been shown in the 60 years since. However, with an SPR

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binding assay, as well as Arabidopsis TIR1/AFB mutants, we were able to revisit the activity of non-

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carboxylic synthetic auxins and this revealed target site specificity more extreme than the known

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prefence of AFB5 for picloram and the picolinate auxins 7, 8. This is the first report of a chemical tool

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for examining receptor-selective responses and redundancy within the TIR1 family of receptors.

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We have suggested the molecular basis of iMT specificity to be a histidine the base of the TIR1

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binding pocket which is replaced by an arginine residue in AFB5 which protrudes more (Figure 6).

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Without the crystal structure of the AFB5 receptor this explanation is based on a homology model 12,

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although this remains a reasonable hypothesis until the AFB5 structure is solved. We recognise that

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we have not represented the AFB2 clade in this study although it clusters close to TIR1 in sequence

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alignments 4. It is clear from all the data collected from tir1-1 plants that the effects of iMT and 4-Cl-

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iMT are dominated by recognition through TIR1. Nevertheless, we can’t exclude some efficacy with

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AFB2 and we note that at the relatively high concentrations needed for activity in vivo with this

245

current generation of iMT analogues there could be some cross-over signalling from other members

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of the TIR1 family. Importantly, we now have chemical templates suitable for TIR1-dominant (iMT)

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and AFB5-dominant (picolinate) activation of auxin signals. We do not yet fully understand the

248

significance of six redundant auxin receptors, but these compound families offer new tools to help

249

differentiate the receptors.

250

The tetrazole bioisostere iMT is not as potent as IAA, with a 40-fold poorer affinity for receptor

251

binding (Table 1). The difference in activity in planta is greater, perhaps due in part to reduced

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uptake and transport. Some reduced uptake capacity is suggested in the timecourse of the protoplast

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gene reporter assays (Figure 4C, Supporting Information Figure 2). Our initial lead optimisation

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programme has yielded increased potency for 4-Cl-iMT with an IC50 value of 19 µM in the primary

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root growth inhibition assay (Table 2). In terms of utility, we may compare this to picloram which is

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a successful commercial herbicide. Picloram has an IC50 of 5 µM (Table 2). A further comparison

257

may be drawn to the herbicide glyphosate, which has an IC50 of 11 mM on its target enzyme 5-


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enolpyruvylshikimate-3- phosphate synthase

34

and an IC50 on Arabidopsis rosettes of 50 µM, with 35

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plants able to grow through treatments at 5 mM .

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As with all agrochemicals, concerns are growing over resistance to current actives. The auxin family

261

of herbicides faces the same challenges. Resistance has not become a global threat 38 39

36, 37

but

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applications are rising with the advent of dicamba- and 2,4-D-tolerant GM crops

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well as maintaining the utility of the current arsenal, binding site variation might open the door to new

264

herbicide selectivities, wider or more restricted than the known broad-leaved dynamic of most current

265

compounds.

266 267


. However, as


268

Methods

269

In silico modelling, chemical and protein visualisation

270

In silico modelling, molecular graphics and analyses were performed with the open source UCSF

271

Chimera package developed by the Resource for Biocomputing, Visualization, and Informatics at the

272

University of California, San Francisco (supported by NIGMS P41-GM103311). 19. Marvin was used

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for drawing, displaying and characterizing chemical structures, substructures and reactions. Calculator

274

Plugins were used for structure property prediction and calculation (Marvin v15.10.12.0, 2015;

275

ChemAxon (http://www.chemaxon.com). Chemical structures were drawn using ChemDraw

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Professional v15.0.0.106. Docking was performed using an automated docking script 21 based on the

277

Vina algorithm 20. Crystal structures 2P1P and 2P1Q 13were sourced from RSCB 40.

278

Recombinant expression:

279

Expression constructs for both TIR1 and AFB5 were engineered to give fusion proteins His10-MBP-

280

(TEV)-FLAG-TIR1 and His-MBP-(TEV)-FLAG-AFB5 respectively and were coexpressed with

281

His10-(TEV)-ASK1 as descrbed in 8. Generation of recombinant virus, quantification, selection,

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expression screening, and generation of high-titer viral stock was done by Oxford Expression

283

Technologies (Oxford, U.K.). Trichoplusia ni (T. ni High 5) was used throughout as the host cell line

284

for expression. Cell densities were determined with a haemocytometer (Marienfeld, Neubauer-

285

improved 0.1mm, catalogue number: 0640030) using a 10x objective lens under a light microscope.

286

Cells were infected at a density of 1 x106 cells/mL with multiplicity of infection of 5. The cells were

287

harvested by centrifugation 72 h after infection and stored at -80oC.

288

Cell Lysis and protein extraction:

289

Frozen TIR1/ASK1 and AFB5/ASK1 pellets were thawed at room temperature and lysed for 40

290

minutes whist rolling at 4oC in Cytobuster™ Lysis medium (Invitrogen 5 mL per 250 mL of cell

291

lysate) supplemented with DNAse I (Roche), protease inhibitors (cOmplete™ Protease Inhibitor

292

Cocktail Tablets, Roche), 50 µM phytic acid (Sigma) and 1 mM reducing agent TCEP (Tris(2-

293

carboxyethyl)phosphine hydrochloride – Sigma). The lysate was diluted upto 30mL into Buffer A (20

294

mM Tris-HCl pH 7.4, 200 mM NaCl, 1mM EDTA, 50 µM phytic acid, 1 mM TCEP) and was

295

subjected to 3 x 30 seconds ultrasonication before centrifugation at 20,000 rpm at 4oC for 15 minutes.

296

The supernatant was then systematically filtered through 0.45 µm and 0.2 µm Whatman GD/X

297

syringe filters.

298 299


Page 10 of 28

Page 11 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300

Protein purification:

301

The filtered lysate was loaded onto a nickel immobilised metal affinity chromatography column

302

(cOmplete His-Tag Purification Resin – Roche), washed with 10 column volumes of Buffer-A before

303

elution with Buffer-B (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1mM EDTA, 50 µM Phytic acid,1

304

mM TCEP, 250 mM Imidazole). TEV protease was added to the elute and incubated with mixing at

305

4oC overnight. The solution was then loaded onto an anti-FLAG-affinity resin (ANTI-FLAG® M2

306

Affinity Gel - Sigma), washed with 10 column volumes of Buffer-C (10 mM HEPES pH7.4, 150 mM

307

NaCl, 3 mM EDTA, 50 uM Phytic acid, 1 mM TCEP, 0.05% Tween 20) and eluted with 3X-FLAG

308

peptide (Sigma) at 100ug/mL. Protein was stored on ice and protein concentrations were assayed by

309

nanodrop A280 nm measurement (Thermo Scientific).

310

SPR assays:

311

The auxin binding assays using SPR were done on a Biacore 2000 instrument as described previously

312

8 41

313

titrating the compounds mixed with constant TIR1 before injection onto the SA chip. The orientation

314

of the assay was otherwise as for the Biacore 2000 assays, and degron peptide density on the chips

315

was controlled so that Rmax > 300 RU.

316

Pull-down assay:

317

The pull down assay was done according to the method described in

318

benthamiana leaves were infiltrated with agrobacterium to express FLAG-TIR1 and leaf lysates were

319

incubated with biotynalted AUX/IAA7 degron (biotinyl-AKAQVVGWP PVRNYRKN) attached to

320

streptavidin beads. The reactions were done in the absence and presence of IAA or iMT at specified

321

concentrations.

322

Plant assays:

323

All root growth assays were done in Col-0 and mutants in this backgroud, tir1-1 and afb5-5 lines 2 27,.

324

A series of plates with 15 serial dilutions for each compound was prepared in half strength Murashige

325

and Skoog medium. From the top concentration of 300 µM a three-fold dilution series was prepared,

326

plus a control without compound, giving a total of 16 plates.

327

From a pool of 6 day old seedlings a random selection of 10 were transferred onto each of the treated

328

plates and the position of the primary root tip was marked. The plates were placed randomly in the

329

stack such that the concentrations were not in order to account for positional bias. A plate with no

330

treatment was included in every assay for comparison with the lower end of the dose response

331

(longest roots) and as an indicator of reproducibility in the assay. The stack was placed with the

. The kinetic analysis of iMT analogues was perfomed by single cycle kinetics on a Biacore T200,


24

where Nicotiana


Page 12 of 28

332

seedlings vertical for 6 days at 12 hour day (22 °C) 12 hour night (18 °C) cycles then scanned (HP

333

PSC 2500 series) at 1200 dpi in colour mode. Primary root growth from the marked point was

334

measured in Image J 42 and plotted using GraphPad Prism v 7.0.

335

The primary root growth measurements were fitted to a logistic function (2.1), using the Levenberg–

336

Marquardt algorithm, in QTI plot (IONDEV SRL, Romania, v 0.9.8.9) . The standard deviation of the

337

data points was weighted into the algorithm = where: M = Maximal value on curve,

338

e = natural logarithm, x0 = IC50 value, k = Steepness of the curve.

339

Lateral root hairs were counted on screen and statistical comparisons were conducted in GraphPad

340

Prism v 7.0 using a two-way ANOVA looking at comparisons within each row (i.e. compound

341

concentration µM) and comparing columns (i.e. mutant lines) to a reference control column (i.e. WT

342

line), multiple comparisons reported to a 95% confidence level (Supporting Information Table 2).

343

DR5::GUS reporter assays:

344

5-days-old DR5::GUS seedlings were cultured in liquid MS medium (1.2% sucrose) with chemicals

345

for 16h at 23ºC. The seedlings were then washed with a GUS staining buffer

346

GUS staining buffer containing 1 mM X-gluc. The seedlings were then incubated at 37 °C until

347

sufficient staining developed.

348

DII::VENUS assay:

349

5-day-old DII::VENUS seedlings

350

yucasin, IAA biosynthesis inhibitor for 6h at 23ºC to accumulate DII-VENUS protein. The chemicals

351

were then added into culture medium at indicated concentration. After 1h incubation at 23 ºC in dark,

352

DII-VENUS image was captured by fluorescent microscopy BX-50 (Olympus, Japan) with YFP filter

353

sets.

354

DR5::GFP reporter assay:

355

Col-0 DR5::GFP seedlings

356

from the plates. Seedlings from ther same batch of germinants were placed onto fresh media (half-

357

strength MS) prepared with compound from 100mM stocks in DMSO to give a final concentration of

358

50µM (final DMSO concentration of 0.05% v/v) in 6-well plates for 2 and 24 h in the dark.

359

Treatments were started simultaneously. At sampling, primary roots were cut to 3 cm, treated with 10

360

ug/ml Propidium Iodide, then placed onto a slide in water and imaged using a Leica (Germany) LSM

361

880 imaging system controlled by Leica Zen software with a 25 x oil immersion objective. GFP was

362

excited with a 488 nm laser line and detected between 499 nm and 544 nm. PI was excited with a 514

363

nm laser and detected between 598 nm and 720 nm. Control roots (DMSO 0.05% v/v) were used to

44

28

43

and transferred to a

were cultured in liquid MS medium (1.2% sucrose) with 20 µM

were germinated as above for root growth assays and used directly


Page 13 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


364

benchmark imaging settings and the same imaging parameters were used for both days, except for the

365

IAA treatment at 24 hours for which the gain was lowered to obtain a non-saturated image.

366 367

Protoplast reporter assay:

368

Mesophyll protoplast were obtained from leaf 8 of 4-week old plants following the “tape sandwich”

369

method using 4 plants per genotype 45. The IAA5 promoter (At1g15580, 920 bp) was amplified from

370

genomic

371

5’CCTGCAGGCTCTAGAGGATCCGCTGTCCATTATCACAAAGTC3’

372

5’TGTTTTTGGCGTCTTCCATGGCTTTGATGTTTTTGATTGAAAG3’). The backbone for the

373

pIAA5::LUC construct was generated from pFRK1::LUC (ABRC CD3-919) by digestion with

374

BamHI and NcoI and gel-purified. Backbone and PCR-amplified IAA5 promoter were combined in a

375

Gibson reaction using the CloneEZ kit (GenScript, USA). Plasmids containing the construct

376

pGH3.3::LUC

377

normalisation

378

concentrations described and LUC substrate luciferin added. The plate was immediately placed in a

379

Photek dark box and imaged with a photon-sensitive camera HRPCS218 (Photek; Supporting

380

Information Figure 4) for 6 h using the software Image32 (Photek) to integrate photon capture. After

381

imaging protoplasts were lysed for GUS activity analysis 46. Images were processed with the Image32

382

software binning the photons captured for each minute into a time resolved image (TRI). Then, for

383

each well total intensity values were extracted. Light intensity was normalised to GUS activity.

384

Quantitative RT-PCR:

385

Leaf number 8 from 4-week old plants was syringe-infiltrated with either 1% DMSO in water (mock),

386

10 µM 4-Cl-iMT or 1 µM IAA for 3 hours. RNAs were extracted with RNeasy Plant Mini Kit

387

(Qiagen) and treated with TURBOTM DNase (Ambion) following manufacturer’s instructions. For

388

cDNA synthesis 1 µg of RNA was reverse-trancribed with the SuperScriptTM II Reverse Transcriptase

389

(Thermo Fisher Scientific), following manufacturer’s specifications, using a primer for polyA tails

390

d(T)19. qPCR was performed with SYBR® Green JumpStartTM Taq ReadyMixTM (Sigma), following

391

the manufacturer’s recommendations (qPCR primers see Supporting information Table 3). All qPCR

392

primers were tested for efficiency on a standard curve. Three technical replicates were used for each

393

sample and 384-well plates were read using a CFX384 TouchTM Real-Time PCR Detection System

394

(Bio-Rad Laboratories). qPCR CT values were exported into an excel file and analyzed using the

395

∆∆CT method

396

stable under the conditions studied (Supporting information Figure 5).

Col-0

46 47

DNA

(primers and

or pIAA5::LUC were transfected together with reporter pUBQ10::GUS for

. After overnight incubation, protoplasts were treated with IAA or 4-Cl-iMT at the

48

. Data was normalised to UBC (AT5G25760;49). UBC expression was found to be



Page 14 of 28

397

Chemical synthesis:

398

iMTsynthesis via 1,3-dipolar cycloaddition

399

yl)acetonitrile with NaN3, AlCl3.THF in THF for 18h at 71˚C (Compound (1), Supporting Information

400

Scheme 1

401

acetonitrile were synthesized according to the published procedure in

402

CL-iMT analougues were synthesised with the NH4Cl, NaN3 into DMF at 120˚C for 30h (Supporting

403

Information Scheme 2). Complete methodologies including NMR and MS data are presented in the

404

Supporting Information (Supporting Information Schemes 1 and 2).

51 52

50

of sodium azide (NaN3) and 2-(1H-indol-3-

. 4-chloroindole-3-acetonitrile, 5-chloroindole-3-acetonitrile and 6-chloroindole-3-

405


53

, their corresponding 4/5/6

Page 15 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


This work was funded by the Biotechnological and Biological Research

406

Acknowledgements:

407

Council BBSRC of the United Kingdom (MIBTP award to MQ, BB/L009366 to RN and SK). We

408

thank M. Estelle for providing the afb5-5 knockout line used in this work. We acknowledge R.

409

Schäfer for invaluable advice in troubleshooting root growth assays and confocal imaging. The

410

protoplast assays were developed under award BB/M017982/1 from BBSRC and EPSRC for the

411

Warwick Integrative Synthetic Biology Centre.

412

List of Supporting Information

413

Scheme 1: Synthesis of iMT

414

Scheme 2: Synthesis of monochlorinated analogues of iMT

415

Table 1: Physiochemical properties of IAA and iMT analogues

416

Figure 1: Additional tetrazoles tested for auxin-like activity

417

Figure 2: Imaging of auxin responses using genetic reporter DII::VENUS

418

Figure 3: Protoplast activity assays

419

Figure 4: Root growth inhibition curves

420

Figure 5: UBC expression observation

421

Table 2: Two-way ANOVA for lateral root densities under 4-CL-iMT

422

Table 3: qPCR reference genes and primer sequences

423

This material is available free of charge via the internet at http://pubs.acs.org

424 425

END OF TEXT

426 427 428 429 430 431 432 433



434 435 436


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437

References

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544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583


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584



Page 20 of 28

Tables:

TIR1 Compound

ka (1/Ms)

IAA iMT 4-Cl-iMT 5-Cl-iMT 6-Cl-iMT

8.06E+02 3.04E+02 2.43E+02 3.28E+02 3.75E+02

± (fit error)

kd (1/s)

3.4 3.2 1.8 8.8 4.7

3.85E-03 6.39E-02 3.82E-02 1.20E-01 7.64E-02

AFB5 ± (fit error)

9.60E-06 1.40E-04 1.90E-04 5.30E-04 2.20E-04

KD (µM)

ka (1/Ms)

4.78 210 157 366 204

4.61E+02 -

± (fit error)

kd (1/s)

6 -

7.02E-02 -

± (fit error)

KD (µM)

4.50E-04 -

Table 1. Kinetic binding data. The Biacore T200 single cycle kinetic facility was used with titrations of each compound against constant receptor concentration to derive kinetic values for each compound.


154 -

Page 21 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A. thaliana lines (IC50 values in µM)

Compound

Col-0

± SE

tir1-1

± SE

afb5-5

± SE

Sensitivity

IAA

0.04

-0.01

0.06

-0.01

0.03

-0.01

-

4-Cl-IAA

0.04

-0.004

-

-

-

-

-

5-Cl-IAA 6-Cl-IAA Picloram iMT 4 Cl iMT 5 Cl iMT 6 Cl iMT

0.19 0.02 4.79 46.21 19.05 59.47 32.48

-0.01 -0.002 -0.87 -2.87 -1.96 -6.85 -1.25

8.4 90.61 36.91 55.04 34.82

-0.84 -6.99 -3.92 -4.29 -2.17

43.66 45.59 19.12 28.09

-6.37 -4.9 -3.39 -1.71

AFB5 TIR1 TIR1 TIR1

Table 2: iMT analogues are active as auxins in the Arabidopsis primary root growth inhibition assay. The IC50 values (±SE) for the inhibition of primary root growth were calculated from statistical fits to dose response data. Receptor preferences are noted on the right.



Page 22 of 28

Figures:

A O

N

R1 OH

R2

NH2

N

Cl

Cl

N H

R3

R4

Structure Name R1 R2 1 IAA H H 2 4-Cl-IAA Cl H 3 5-Cl-IAA H Cl 4 6-Cl-IAA H H

N H

R2

N H

R3

N

R1

R4 H H H H

Structure Name 5 iMT 6 4-Cl-iMT 7 5-Cl-iMT 8 6-Cl-iMT

OH

N O

R4

R3 H H H Cl

Cl

9 R1 H Cl H H

R2 H H Cl H

R3 H H H Cl

B

R4 H H H H

Figure 1. Auxin analogues Chemical structures: of IAA (1) and iMT (5) with their respective mono-chlorinated analogues, and the synthetic auxin picloram (9).


Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B

IAA

iMT

SER438

SER438

3.47Å

1.89Å

4.52Å

2.34Å ARG403

ARG403

ARG436

ARG436

C

D

SER438

2.3Å 2.7Å 3.6Å

1.9Å

ARG403 ARG436

ARG436

ARG403

Figure 2. Modelling iMT as a ligand for TIR1. A: The crystal structure of IAA (gold) bound in the TIR1 receptor, with key residues shown with coloured heteroatoms. Interatomic distances are shown as dashed black lines. B: As A, but with the tetrazole analogue modelled in the binding site using Chimera. C: IAA (magenta) and iMT (grey), each docked (AutoDock Vina) in the deep binding pocket of TIR1 (gold, ribbon). The docked poses are consistent with the modelling in B. The indole ring of iMT adopts the same plane as that for IAA (which is the same as that seen in the crystal structure), with the tetrazole group projecting past the carboxylic acid group of IAA. D: shows the atomic distances between the tetrazole group nitrogens and neighbouring arginine residues.



Figure 3. iMT binds to TIR1, but not to AFB5. SPR binding curves for compounds screened at 50µM against TIR1 (Panel A) and AFB5 (Panel B). In each case IAA (red trace) is used for reference. With TIR1 we observe a saturating binding response with a rapid off rate for all iMT analogues. None of the iMT analogues were active against AFB5. C: A pull-down assay for FLAG-TIR1 in the presence of increasing concentrations of IAA (left) and iMT (right). A western blot developed with anti-FLAG antibody detects FLAG-TIR1 (arrow) bound to streptavidin-coated beads loaded with biotinylated degron peptide. As with SPR, there is a strong response with IAA, and the iMT response is dose-dependent, but weaker.


Page 24 of 28

A

Page 25 of 28

A B

Mock

B C

Mock DMSO

IAA 1µM

iMT 50µM

4-Cl-iMT 50µM

5-Cl-iMT 50µM

6-Cl-iMT 50µM

Mock

IAA IAA 1µM 1µM

iMT iMT 50µM 50µM

4-Cl-iMT 4-Cl-iMT 50µM 50µM



IAA IAA 1µM

iMT iMT 50µM

4-Cl-iMT 4-Cl-iMT 50µM



iMT

4-Cl-iMT

5-Cl-iMT

6-Cl-iMT


100 µm

2 hours

B

24 hours

*

C

DMSO

100 µm

IAA

2 hours

C

100 µm

pIAA5::LUC

Relative24 light intensity hours

IAA 600

4-Cl-iMT 600

*

400 200

pGH3.3::LUC

30 µM

200

1 µM

0 100 200 300 Time [minutes]

10 µM Mock

Mock

0

400

1500 Relative light intensity

400

100 µM 10 µM

0

0

100 µm

100 200 300 Time [minutes]

400

1500

1000

100 µM

1000 30 µM

10 µM

500 0

500

1 µM Mock

0

100 200 300 TIme [minutes]

0

400

10 µM Mock

0

100 200 300 Time [minutes]

400

D IAA5

GH3.3

15

5 0

GH3.3

30

10

WT WT Col-0

GH3.1

150

30

Fold change

Fold induction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Mock

20 10

20

100

10

50

0

tir1-1 afb5-5 afb5-5 tir1-1

Mock

0

WT WT WT Col-0

tir1-1 afb5-5 tir1-1 tir1-1afb5-5

10 µM 4-Cl-iMT

0

WT tir1-1 tir1-1 afb5-5 afb5-5 WT afb5-5 Col-0

1 µM IAA

Figure 4. iMT analogues are active auxins in vivo. A: The DR5::GUS reporter line indicates auxin activity of iMT and its monochlorinated analogues. Treatments were for 16 h at 1µM for IAA, 50 µM for iMT and its analogues. No activity was seen with 5-Cl-iMT. B: Signals from DR5::GFP after 2 and 24 hours of treatment. Activity is seen after 2 hours with IAA, iMT and 4- and 6-Cl-iMT show activity within 24 hours, with 4-Cl-iMT giving a comparatively strong response. *For the image of IAA treatment after 24 hours the gain was lowered to avoid a saturated signal. C: Arabidopsis protoplasts were transformed with auxin-sensitive reporter constructs pGH3.3::LUC (above) or pIAA5::LUC (below) before treatment with IAA (left) or 4-Cl-iMT (right), each over responsive dose ranges and each with a mock treatment (blue). Luciferase activity was recorded each minute for 6 hours. Error bars represent standard deviations of technical replicates and the plots are a representative set from three biological repeats. D: Quantitative PCR data for three auxin-responsive reporter genes, IAA5, GH3.3 and GH3.1. RNA samples were prepared from treated leaf tissues collected 3 hours after treatment with mock, 4-Cl-iMT or IAA (10 and 1 µM respectively) from Col-0, tir1-1 and afb5-5 lines. Error bars represent the standard error of the mean of biological repeats (n = 4).



A tir1-1 100

IAA

4-Cl-iMT

Concentration (µM)

5 01

04 0.

Low

0.

6

0

4

5

6

0. 01

0. 04

1

0. 4 0. 13

3

11

0

33

0

10

30

0. 04 6 0. 01 5

1

0. 4 0. 13

3

11

0

33

0

10

30

Concentration (µM)

20

6-Cl-iMT

0

0

13

6-Cl-iMT

6-Cl-iMT

40

5-Cl-iMT

20

0.

5-Cl-iMT

60

1

20

Picloram

40

0.

5-Cl-iMT

60

4-Cl-iMT

3

40

11

4-Cl-iMT

iMT Picloram

0

60

High

80

iMT

0

Picloram

100

IAA

80

30

iMT

80

33

100

IAA

Lateral root density

afb5-5

10

Col-0 WT

Concentration (µM)

B

Number of laterals per cm of root

20 ****

15 **

10

5

WT

02 0. 05 0. 14 0. 41 0. 23 1. 70 3. .11 11 3 .3 33 00 0. 10 0 0 0. 30

Col-0

02 0. 05 0. 14 0. 41 0. 23 1. 70 3. .11 11 3 .3 33 00 0. 10 .00 0 30

0

Untreated control range 02 0. 05 0. 14 0. 41 0. 23 1. 70 3. .11 11 3 .3 33 0 0 0. 10 0 0 0. 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

Concentration (µM)

tir1-1

tir1-1

afb5-5

afb5-5

Figure 5: The tir1-1 mutant is insensitive to iMT and 4-Cl-iMT A: Normalised Lateral root densities plotted as heat maps to display the effects of compound and compound concentration for wild type, tir1-1 and afb5-5 knockout lines. Lateral root density is reduced in the tir1-1 mutant challenged with iMT, 4-Cl-iMT and 6-Cl-iMT, but not in afb5-5 nor Col-0. Cross-hatching (XX) indicates that this was not tested. B: From the same data as A but focussing on lateral root densities for lines treated with 4-Cl-iMT. Error bars indicate +/- the standard error of the means. A two-way ANOVA was used to compare densities at each concentration for each mutant line versus Col-0; ** P≤0.01 and **** is for P≤0.0001 using 10 replicates per concentration per seed line. The loss of density at higher concentrations is seen only with tir1-1 and not in the afb5-5 line.


Page 27 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A A

Protein TIR1 Protein Protein' AFB5

77

78

79

80

81

82

83

84

P H F A D F N L

344 345 346 347 348 349 350 351 352 353 354

377 378 379 380 381

R V F P S E P F V M E

V L Y F C

TIR1

R F

122 123 124 125 126 127 128 129 391 392 393 394 395 396 397 398 399 400 401 423 424 425 426 427 77" 78" 79" 80" 81" 82" 83" 84" 344"345"346"347"348"349"350"351"352"353"354" 377"378"379"380"381" 401"402"403"404"405"406"

P R F A D F N L

R

I

F P F D P R E D S

R L C

R L S L S G

I

L Y F C

"TIR1" P" H" F" A"D" F" N" L" " R" V" F" P" S" E" P" F" V"M E"" V" L" Y" F" C"" R" F" R" L" C" I" "AFB5" R" F" A"D" F" N" L" " R" I" F" P" F" D" P" R" E" D" S"" I" L" Y" F" C"" V" F" R" L" C" I" Protein P" 403 404 405 406 407 408 409 410 436 437 438 439 440 441 460 461 462 463 464 465 485 486 487 I

405"406"407"408"409"410" Protein' Protein 77"

I

E P K

M L S V A F

436"437"438"439"440"441" 460"461"462"463"464"465" 485"486"487"488"489"490" 449 450 451 452 453 454 455 456 482 483 484 485 486 487 506 507 508 509 510 511

"TIR1" L" C" P"I"R I" LE" CP" K"" I" R"V D"A AFB5 I R" M L" G S"RL" HS" G"" RM L L"AS" VV" A" S F"" G K"TL" LE" S "AFB5" L" C" P"I" M G" R" H"" R" L" A" V" S" G"" T" L" S" V" A" F"" K" L" E" I" R" D"

F

K L E

401 402

447 448

V F

488 489 490

I

R D

531 532 533 534 535 536

K L E

I

R D

B

C ARG123 ALA484

ARG123 ALA484 ARG449 ARG449

D

E ARG123 ALA484

ARG123 ALA484 ARG449

F

ARG449

G ARG123 ALA484

ARG123 ALA484 ARG449

ARG449

Figure 6. A model for iMT selectivity based on space constraints in AFB5. A: The binding pocket residues of TIR1 are alligned against those of AFB5, using the residue numbers for TIR1 (Clustal 2.1). Key differences are highlighted in yellow and include His78, which is Arg in AFB5, and S438 Ala in AFB5. B – G: Views of the homology model for AFB5 (Calderon-Villalobos et al., 2012) showing IAA (B and C) or iMT (D and E), or both F and G) docked using AutoDock Vina. The views in each left hand panel are similar to those for TIR1 in Figure 1, and on the right views are revolved to show the pose of the side group out of the plane of the aromatic ring system. Note that iMT does not adopt the same pose as IAA in AFB5 because the space occupied by the tetrazole in TIR1 (Figure 1) is partially occupied by Arg123 in AFB5, forcing the tetrazole up and away from the base of the pocket, resulting in the indole twisting from alignment with the base of the pocket.


ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Page 28 of 28

N N N N H

O OH

Cl NH

NH TIR1 binding AFB5 binding

TIR1 binding

! !!"

AFB5 binding

! !!"


! !!"

The tetrazole analogue of the auxin indole-3-acetic acid binds

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